Establishment And The Research Of Chronic Colitis Animal Model In BALB/C Mice Induced By TNBS

Background: Crohn's disease (CD)-associated fibrosis results from chronic transmural inflammation. Intestinal inflammation in CD is transmural, often associated with extracellular matrix changes, luminal narrowing and stricture formation. But the knowledge of stricture formation pathogenesis remains unclear. Current therapies do not alter its progression to intestine fibrosis and obstruction. The need for simple, effective, good reproducibility animal model like human Crohn's disease is urgent.Objective:To explore the feasibility of chronic colitis animal model in BALB/C mice induced by six weeks TNBS/ethanol enemas. Observe the pathological changes and the level of various types of collagen, TNF-α, TGF-βof colon tissue. Study the difference feature between acute and chronic colitis animal model induced by TNBS, the possible pathogenic mechanisms and treatment target.Method: BALB / C mice which fasted but drunk for 24h before each TNBS/ethanol enema. They were randomly divided into blank control group, 50% ethanol control group, TNBS-induced acute colitis model group, TNBS-induced chronic colitis model group I, II. each group had 12 of them. The mice in blank control group were administered six weeks with only 100ul saline enema and killed in 7th week. The mice in 50% ethanol control group were administered six weeks with only 100ul ethanol (50%) enema and killed in 7th week. The mice in TNBS-induced acute colitis model group were administered 2.0mg TNBS/ethanol (50%) enema only one week, killed in 2nd week. The mice in TNBS-induced chronic colitis model group I, II were administered six weeks with TNBS/ethanol (50%) enema, the group I killed in the 7th week, the group II killed in 9th week. Colon tissue were collected for the following experiments , which included inflammatory scores and mucosa morphological changes under light microscopy, the level of various types of collagen, TGF-β, TNF-αmRNA in colon tissue examined by RT-PCR, the protein of TGF-β1, TNF-αdetected by immunohistochemistry.Result:1. After the administration of the first dose of TNBS the mice show different symptoms in TNBS-induced chronic colitis model group I, II, However, during the course of the next 4-5 wk, the surviving mice gradually regained lost weight and recovered from other obvious signs of chronic illness despite continued TNBS administration. Compared with blank control group and 50% ethanol control group, the DAI score increased in TNBS-induced acute colitis model group, TNBS-induced chronic colitis model group I, II (P<0.05), but DAI score declined in TNBS-induced chronic colitis model group I, II compared with TNBS-induced acute colitis model group (P<0.05).2. Severe inflammation can be detected through naked eye and microscope in mice of TNBS-induced acute colitis model group, like the lamina propria of the colon was characterized by a massive infiltration of inflammatory cells, and evidence of epithelial cell disruption. Compared with other groups, the inflammation score increased in TNBS-induced acute colitis model group (P<0.05). Obvious collagen deposition in the subepithelial area, smooth muscle area and in the serosal area of mice in TNBS-induced chronic colitis model group I, II, especially in the proximal colon. The inflammation score in TNBS-induced chronic colitis model group I, II increased only compared with blank control group (P<0.05), but fibrosis score increased compared with other groups (P<0.05).3. The level of Col-Iα2,Col-IIIα1,Col-Vα2 mRNA in TNBS-induced chronic colitis model increased compared with other groups, TGF-β(P<0.05), TNF-αmRNA in TNBS-induced acute chronic colitis model increased compared with other groups (P<0.05).4. The level of TGF-β1 in TNBS-induced acute colitis model group, TNBS- induced chronic colitis model group I, II increased compared with blank control group and 50% ethanol control group (P<0.05), but declined in TNBS-induced acute colitis model group I, II compared with TNBS-induced chronic colitis model group I, II (P<0.05). The level of TNF-αin TNBS acute colitis model group, TNBS-induced chronic colitis model group I, II compared with others (P<0.05), no statistical difference in them.Conclusion:1. We had established acute and chronic colitis animal model induced by TNBS successfully which determined by the DAI and pathological changes.2. The pathological features of colon tissue is acute inflammatory in TNBS-induced acute colitis model group, and chronic inflammatory TNBS-induced chronic colitis model group, Obvious collagen deposition can been seen in the subepithelial area, smooth muscle area and in the serosal area of mice in TNBS-induced chronic colitis model group I, II, especially in the proximal colon. The characteristics of intestinal inflammation and fibrosis in two models are same to the CD, respectively, reflect the a acute inflammation stage and chronic fibrosis Phase in CD.3. The TNBS-induced chronic colitis model features chronic intestinal inflammation combined with abundant collagen deposition and fibrogenic ECM changes, it is stable for 2 weeks after TNBS treatment.4. Acute injury may cause normal mesenchymal cells to convert to fibrogenic phenotype that may lead to fibrosis when inappropriately sustained. The over- expression of TGF-β1 play an important role in the fibrosis process, it could be the treatment target of intestinal fibrosis in CD.5. TNF-αincreased in TNBS-induced acute, chronic colitis model, The reason is that TNBS as hapten protein, activates antigen-induced immune response, or TNF-αas a fibrosis-related factors involved in the fibrosis process. So, TNF-αplay an important role in the process of intestinal inflammation and fibrosis.